Comprehensive Profiling of Four Base Overhang Ligation Fidelity by T4 DNA Ligase and Application to DNA Assembly

Abstract

Synthetic biology relies on the manufacture of large and complex DNA constructs from libraries of genetic parts. Golden Gate and other Type IIS restriction enzymedependent DNA assembly methods enable rapid construction of genes and operons through one-pot, multifragment assembly, with the ordering of parts determined by the ligation of Watson−Crick base-paired overhangs. However, ligation of mismatched overhangs leads to erroneous assembly, and low-efficiency Watson Crick pairings can lead to truncated assemblies. Using sets of empirically vetted, highaccuracy junction pairs avoids this issue but limits the number of parts that can be joined in a single reaction. Here, we report the use of comprehensive end-joining ligation fidelity and bias data to predict high accuracy junction sets for Golden Gate assembly. The ligation profile accurately predicted junction fidelity in ten-fragment Golden Gate assembly reactions and enabled accurate and efficient assembly of a lac cassette from up to 24-fragments in a single reaction.

Synthetic DNA Assembly Using Golden Gate Cloning and the Hierarchical Modular Cloning Pipeline

“…2D). The restriction-ligation leads to a high cloning efficiency and allows up to 24 fragments to be ligated in a single cloning reaction (Potapov et al, 2018).…”

“…The fusion site needs to be different from the other fusion sites (AATG and GCTT) and should not be palindromic. It should contain at least one G or C (more, if possible), as sites containing only As or Ts may work less well for DNA assembly (Potapov et al, 2018). When many fusion sites are needed, the Ligase Fidelity Viewer program (New England Biolabs, http:// ggtools.neb.com/ viewset/ run.cgi) can be used…”

Effective Characterization of DNA Ligation Kinetics by High‐Resolution Melting Analysis

“…Ligation in the presence of T4 DNA ligase involves a complicated mechanism, and no kinetic data on its connection of sticky ends have been available (only nick sealing in the presence of this enzyme has previously been analyzed, with a DNA sequencer and a stopped‐flow machine) . Here, k obs and apparent K m values for the ligation of DNA1/DNA2 (full match) and G‐G mismatch were determined by HRM analysis (Figure ).…”

An all-to-all approach to the identification of sequence-specific readers for epigenetic DNA modifications on cytosine

“…To carry out the DAPPL reactions, the 20-mer DNA library (~1 X 10 12 species) was incubated with the barcoded TF mixtures, followed by stringent washing steps to remove unbound DNA. After crosslinking, a Golden Gate Assembly reaction (Potapov et al, 2018) was performed to ligate the DNA barcodes of the TFs to their captured DNA fragments. The ligated DNA products were PCR-amplified with specific primers to construct libraries for deep sequencing (Figure 1).…”